EP1808689B1 - Neue Verfahren zur Immunozytfärbung zur verbesserten Unterscheidung des Färbungsverhältnisses bei der Erkennung seltener Ereignisse - Google Patents

Neue Verfahren zur Immunozytfärbung zur verbesserten Unterscheidung des Färbungsverhältnisses bei der Erkennung seltener Ereignisse Download PDF

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EP1808689B1
EP1808689B1 EP07100507A EP07100507A EP1808689B1 EP 1808689 B1 EP1808689 B1 EP 1808689B1 EP 07100507 A EP07100507 A EP 07100507A EP 07100507 A EP07100507 A EP 07100507A EP 1808689 B1 EP1808689 B1 EP 1808689B1
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Prior art keywords
antibody
marker
sample
type
laser
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French (fr)
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EP1808689A1 (de
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Ben H. Hsieh
Nicole Hanson Lazarus
Robert T. Krivacic
Douglas N. Curry
Richard H. Bruce
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SRI International Inc
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SRI International Inc
Stanford Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6482—Sample cells, cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6484—Optical fibres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/08—Optical fibres; light guides
    • G01N2201/0833—Fibre array at detector, resolving

Definitions

  • the present application relates to laser based detection systems, and finds particular application in conjunction with low and high-density cell detection and discrimination in blood smears, biological assays, and the like, and will be described with particular reference thereto. However, it is to be appreciated the present concepts will also find application in detection and discrimination of other types of low- or high-density features on various substantially planar surfaces and samples.
  • Laser based detection systems are widely used in many industries, including printing, bio/life and medical sciences, and are implemented in biochip readers, and laser scanning cytometers, among other detection systems.
  • laser light is guided through objectives similar to those for microscopes. These objectives utilize multiple lens elements to achieve high magnification and often near- or sub-micron resolution. Since both excitation and emission light are guided through these objectives, the heavy weight of the objectives and their small aperture limits the speed at which the laser light can be moved and thus limits the speed of scanning of a sample.
  • FAST Fiber Array Scanning Technology
  • PARC Palo Alto Research Center
  • FAST employs a rapid spinning galvanometer or mirror for directing laser light and a large-aperture fiber bundle to collect light emission over a relatively large area.
  • the FAST scanning speed is very high, however, its spatial resolution is currently at a laser spot of approximately 8 ⁇ m.
  • Patent Applications 2004/0071330 Imaging Apparatus and Method Employing a Large Linear Aperture
  • 2004/0071332 Apparatus and Method for Detecting and Locating Rare Calls
  • the prepared sample is configured to emit signals having spectral characteristics sufficient to permit filtering to differentiate and eliminate most false positives from true positives among acquired imaging events, in an imaging system employing a laser spot having a range of diameters from 1 to 20 ⁇ m or greater and which excites the fluorescence in a conventional or novel manner. These filtered events may be subsequently imaged and confirmed with another higher resolution device such as a fluorescent microscope in a short amount of time.
  • the present invention includes a method of subjecting a sample potentially containing cells of interest to a laser scanning process as defined in claims 1 to 10.
  • Imager 10 employing the Fiber Array Scanning Technology (FAST), is depicted.
  • Imager 10 examines a sample 12 with biological smear 14 disposed on at least a portion of a surface of a slide 16.
  • FAST Fiber Array Scanning Technology
  • sample 12 generating a fluid or a smear, is prepared e.g. by drawing a sample of a biological fluid such as, but not limited to, blood or parts of blood from a subject.
  • a biological fluid such as, but not limited to, blood or parts of blood from a subject.
  • the sample is a monolayer of cells adhered to a slide,in particular, of blood cells with red cells removed.
  • the fluid sample is treated with a fluorescent material, such as but not limited to a biological marker conjugated to dye fluorophore that selectively bonds to a biological molecule, which may be on the surface or inside a cell, such as a protein, nucleic acid or other molecule.
  • Suitable markers are known in the art for marking a number of different cell types of clinical interest, including selected cancer cell types, fetal cells, or other appropriate cells to be considered. Work is also being undertaken to develop markers for numerous cell types from other organs such as brain cells, liver cells, as well as for bacterial cells or viruses, among others.
  • the marker preferably emits a characteristic luminescing output, such as a fluorescence or phosphorescence light, responsive to a selected excitation irradiation, such as irradiation by a selected wavelength or spectrum of light, x-ray irradiation, electron-beam irradiation, or the like.
  • the characteristic luminescence typically has a characteristic wavelength or spectral range of wavelengths. While organic dyes (i.e. fluorophores) are the predominant markers, other marking techniques exist including the use of other markers known as quantum dots and nano-particle probes. Systems using these as well as other materials and techniques may beneficially employ the concepts of the present application.
  • the sample e.g. smear
  • size will depend on implementation, however.
  • the smear 14 might contain 10 6 to 50 x 10 6 or more cells and occupy an area of about 10 cm 2 to 100 cm 2 or greater.
  • larger or smaller smears can be prepared which are suitable for the anticipated concentration of cells in the sample and the desired minimum measurable cell concentration.
  • Sample 12 is mounted on an imager translation stage 18 (shown in a partial view) which includes a linearly translatable track 20 that supports sample 12.
  • a motor 22 connects with track 20 via gearing 24 to translate track 20 and the supported sample 12 along a ⁇ -direction (indicated by arrows 28) in an x-direction (indicated by arrows 28).
  • a light pipe 30, such as a fiber optic bundle includes a first end 32 that is proximate to the sample 12, and a second end 34 that is distal from the sample 12.
  • the first end 32 includes a plurality of first fiber ends arranged substantially parallel to one another in an arrangement that defines a generally linear or high-aspect-ratio rectangular input aperture 36 with a long dimension aligned with the x-direction.
  • the optical fiber bundle 30 changes cross-sectional dimensions and shape between the first end 32 to the second end 34 such that the second end 34 includes a plurality of second fiber ends that define a compact, generally circular, output aperture 38.
  • a scanning radiation (light) source 40 in a suitable embodiment includes a laser 42 that produces excitation light (radiation beam) 44 at a wavelength or wavelength range selected to excite the marking material used in marking the biological smear 14.
  • the excitation light 44 is angularly scanned by a galvanometer 46 that has a reflective surface that rotates (indicated by curved arrows 48) responsive to an electrical input.
  • An optional focusing lens 50 focuses the angularly scanned excitation light 64 onto the sample 12, and more particularly onto the biological smear 14.
  • the angular scanning produced by the galvanometer 46 translates into a linear sweeping or fast scanning (indicated by arrows 52) of the excitation light, preferably in the form of a spot, which presently is approximately 8 ⁇ m or greater in diameter on the biological smear 14 along a linear trajectory 54 arranged below the input aperture 36 and parallel to the long dimension of the input aperture 36.
  • An electronic control unit 56 communicates with the galvanometer 46 and the translation stage 18 to coordinate the linear sweeping or scanning 52 of the radiation beam 44 along the trajectory 54 and the linear translation 26 of the sample 12 to effectuate a rastering of the radiation beam 44 across a selected area of the sample which is bounded in the x-direction by the smaller of a span of the trajectory 54 and the long dimension of the input aperture 32.
  • the span of the trajectory 54 substantially comports with the long dimension of the input aperture 32.
  • the scanning radiation source 40 and the input aperture 36 are arranged in fixed relative position, the galvanometer 46 provides a linear sweeping of the excitation beam 44 along the x-direction, and the sample 12 is moved by the translation stage 18 linearly along a y-direction to effectuate a two dimensional rastering.
  • a suitable signal detector 58 is arranged to detect the collected characteristic luminescence emanating from the output aperture 38.
  • a first lens 60 substantially collimates the light.
  • Blocking filter 62 is optionally provided to remove scattered laser light from the collected light.
  • a second lens 64 may be provided to focus the collimated collected light onto a photodetector arrangement 66.
  • a photodetector arrangement 66 Combining the compact output aperture 38 with focusing optics 60, 64, photodetector 66, which may be a single photodetector, provides signal detection for the spatially distributed linear input aperture 36.
  • the photodetector 98 is preferably a photomultiplier tube.
  • Electronic control unit 56 communicates with the galvanometer 46 and the translation stage 18 to raster the radiation beam 44 across the sample. Characteristic luminescence produced by interaction of the radiation beam 44 with treated cells in the biological smear 14 is collected by the input aperture 36, channeled to the output aperture 38 by the optical fiber bundle 30, and detected by the signal detector 58. The electronic control unit 56 receives the detected signal from the photodetector 66, and correlates the detected signal with positional coordinates of the radiation beam 44 on the sample 12.
  • the electronic control unit 56 suitably formats the detected signal and spatial coordinates information and stores the information in an internal memory, writes the information to a non-volatile storage medium such as a magnetic or optical disk, formats and displays an image representation including an array of picture elements with coordinates mapped to the spatial coordinates information and an intensity or color mapped to the detected signal intensity on a display 68, or the like.
  • the markers As previously discussed, during the scanning operations, interaction of the spot generated by the laser beam with tagged cells in a sample will cause the markers to emit a luminescence, such as a fluorescent light. Commonly, these markers are clustered within the cells and generate high-intensity pixels when they are excited and emit luminescence upon scanning by the radiation spot. For the following discussion, the detected unknown cluster of markers is described as an "image event" to which further investigation is warranted.
  • the size of the radiation spot defines the resolution of the imaging device.
  • the imager 10 may accumulate image data irrelevant to the identification of rare cells. At times this noise may be detected as "false positives.” It is desirable to eliminate this noise during image acquisition and processing. Therefore, filtering procedures are implemented via electronic control unit 56 and/or other elements of system 10 to eliminate information not related to rare cells.
  • the filtering techniques may use various characteristics of an image event to perform the filtering operations, including the number of pixels, intensity, phase and shape of the image event under consideration.
  • an image event may be classified as a non-rare cell (false positive) or a rare cell (true positive) image event by counting the number of pixels of the image event under investigation. Knowing approximate of rare cell marker clusters under investigation, a range can be set to filter out those image events having either a number of pixels less than or greater than the prescribed range. For instance, if a rare cell would be known to correlate to a number of pixels in a range of 1 to 12, then image events having a pixel range greater than 12, would be eliminated in a filtering operation.
  • the shape of an image event is used to filter non-relevant information.
  • an image event correlating to a rare cell or cluster of rare cells would have a known shape corresponding to the rare cells being imaged, and blurred by the impulse response of the radiation spot. If the detected shape is other than expected for the pertinent rare cell and/or clusters of rare cells, this would indicate the detected image event is noise such as a dust or dirt particle or other irrelevant signal from the sample.
  • known pattern matching software may be implemented in imaging system 10. In this filtering operation, it would be expected not to see an image event that had a finer structure than the spot's own resolution size, Particularly, the image event would not be smaller than the spot size, although the structure itself may be smaller.
  • Still a further filtering process which may be used to identify rare cell image events from non-rare cell image events is by tracking the intensity of the image event under investigation. For example, it would be expected that a higher intensity would be detected for rare cell image events that are in phase with the pixel acquisition phase, and would also provide fewer pixels. Out of phase image events would have their energy shared with several neighboring pixels, thereby providing a smaller intensity per pixel, but more pixels. In addition, in some non-specific binding of tags on cells, i.e., cells not related to the rare cells, may produce image events but these would have a lower intensity than the expected intensity from rare cell binding clusters.
  • filtering techniques which may be used to screen for rare cell events (true positives) from the image events detected by the imaging system.
  • a manner to address the issue of insufficient spatial resolution in a large view/fast scanning system, such as FAST, is to improve sample preparations in a manner whereby during the detection procedure spectrally distinguishable characteristics of the sample that are unique to true positive cells are emitted.
  • false positives may be minimized in a FAST based type system by improving the spectral characteristics of the prepared samples, to permit easier discrimination between image events.
  • broadband excitation sources e.g. , high pressure mercury arc lamp, xenon lamp
  • bandpass or longpass filters optimized for exciting specific fluorophores (or dyes), in particular in multiple-color microscopy application.
  • the fluorphore can be pre-conjugated to primary antibody so that when put together they bind to the cellular target (antigen), the whole assembly fluoresces when properly illuminated.
  • a fluorophore-conjugated secondary antibody can be used; this secondary is immunized with the serum from the host in which the primary is raised, so it will specifically target the primary antibody.
  • Using the secondary antibody has the advantage of a brighter signal due to signal amplification because multiple copies of the secondary antibody can bind to a single primary antibody, thereby providing for increased ease of discrimination of image events.
  • the laser spot size is usually in the range of a few to tens of microns.
  • the spot size is approximately 8 ⁇ m or greater in diameter, which is close to the size of a human blood cell ( ⁇ 10 ⁇ m) and a typical occult circulating cancer cell. It therefore is not able to provide sub-micron resolution needed to view cellular structural characteristics.
  • using raster scanning with a galvanometer in a FAST imager achieves high speed, it is at the expense of this lower resolution. The resolution is not sufficient to distinguish whether a particular image event represents a false positive which may be an auto-fluorescent artifact, dye aggregate or a true positive, i.e. a genuinely labeled cellular target.
  • an anti-cytokeratine primary antibody may be used at 1:100 dilution to target potential epithelial (likely cancerous) cells circulating in the blood stream, with a secondary antibody such as Alexa 488-conjugated goat anti-mouse Ab at 1:1000 dilution to identify the targeted cells.
  • a secondary antibody such as Alexa 488-conjugated goat anti-mouse Ab
  • thousands or more potential hits or image events may be registered at the regular emission band of Alexa 488, i.e. 525nm +/- 20nm. However, only a few or perhaps none may be true positives.
  • improved spectral characteristics of a sample may be achieved by using unconventional excitation and emission methods to eliminate background fluorescence and by employing multiple markers (e.g., dyes, etc.) in various ways that target the same cells to create emission ratio signatures that uniquely identify true positives.
  • An unconventional excitation and emission includes employing a laser and a marker together which does not result in an optimal fluorescing of the marker.
  • the examples refer to the marker as a fluorophore dye, however it is to be understood other appropriate markers are also appropriate for use in connection with the present concepts.
  • FIGURE 2 instead of using just one fluorophore conjugated (i.e ., associated) to the secondary antibody, two fluorophores are used.
  • this procedure illustrates a primary antibody (1 st Ab) 70 which is bound to a cellular target 72 of interest, by a known technique.
  • the cellular target is part of material having been placed on a slide with a sample such as in FIGURE 1 .
  • multiple secondary antibodies 74 (of a same type) having two different versions of fluorphores 76(*), 78(+) are selectively conjugated thereto. Both the conjugated fluorophores 76, 78 are designed to target the same primary antibody 70.
  • the emission intensity of the two fluorophores reflects the concentration used.
  • Alexa 488 goat anti-mouse Ab and Alexa 555 goat anti-mouse Ab may be used to target and compete for primary mouse anti-human cytokeratin Ab.
  • Table 1 which lists quantum properties of selected Alexa dyes, and as illustrated in FIGURE 3 , which depicts excitation and emission curves of the Alexa dyes 488 and 555, when a 488nm laser is used, Alexa 488 is excited at much higher efficiency (75.1 % (488nmEx. Efcy.) according to data from Molecular Probe/Invitrogen Corporation of Carlsbad, California) than Alexa 555 (14.2%).
  • FIGURE 3 the excitation and emission spectra of Alexa dyes (EX_488 (80), EX_555 (82), EM_488 (84), and EM_555 (86)) are plotted by absorbance or emission efficiency (Percent Efficiency) versus wavelength (Wavelength (nm)).
  • Table 1 Alexa488 Alexa555 Ex Peak (nm) 499.0 553.0 Em Peak (nm) 520.0 568.0 Exit.Coef. 7.1E+04 1.5E+05 488nm Ex. Efcy (%) 75.1 14.2
  • Alexa 555 e.g ., 16
  • Alexa 488 antibody e.g . 14
  • Alexa 555 (16) is provided at a concentration ratio of 100:1 as compared to the Alexa 488 (18) antibody.
  • Alexa 555 emission intensity and/or the amount of emitted photons that can be collected
  • the 488nm laser such as laser 42 of FIGURE 1
  • concentration ratios may be applied, in order to create the asymmetric marker arrangement, which provides the desirable spectral resolution.
  • blood autofluorescence would typically have an emission peak around that of Alexa 488 ( ⁇ 525nm) when excited by a 488nm laser, and very little or none at that of Alexa 555 ( ⁇ 580nm).
  • Testing of various cancer cell lines when excited by one 488nm laser have shown that virtually all of determined true positives have emission intensity ratios (580nm vs. 525nm; in the form of average intensity per pixel) greater than 1.0, with the majority the true positives at 2 or above, when the Alexa 555 dye is used at 100:1 aver Alexa 488 dye. The majority of false positives were determined to have a ratio below 1.0.
  • Alexa 555 Although the above asymmetric concept provides improved detection and elimination of false positives, there are some issues associated with this procedure. Particularly, the low excitation efficiency of Alexa 555 by a 488nm laser, combined with a bleeding of Alexa 488 emission to Alexa 555 emission area ( ⁇ 580nm, see the tail of Alexa 488 emission, e.g., EM_488 (84) in Figure 3 ) requires the use of a substantially high enough amount of the Alexa 555. Furthermore, in order to avoid a weak signal and therefore non-detection, Alexa 488 concentration cannot be too much lower than a regular or commonly used 1000x dilution.
  • Alexa 488 may be used at a 3000x dilution while Alexa 555 at 30x dilution to substantially ensure a high probability that all true positives have an emission ratio >1.0.
  • a 30x dilution of a typical secondary antibody stock e.g. 2mg per mL
  • a second issue in the asymmetric implementation is that the competitive nature of two secondary antibodies 76, 78 binding to single primary antibody 70 means the results of such bonding can sometimes be less than consistent across different experiments, especially in cells with a low antigen expression level, as the primary antibody can be easily saturated.
  • FIGURE 4 depicted is a second approach used to improve the spectral distinctiveness of a sample.
  • one (or a first) fluorophore 90(+) is conjugated to a primary antibody 92, which is bound to a cell of interest 94.
  • Another (or second) fluorophore 96(*) is conjugated to a secondary type antibody 98, selected and prepared to target the fluorophore-conjugated primary (or 1 st type) antibody 92.
  • this binding is sequential and non-competitive and can be allowed to go to completion.
  • Alexa 488 mouse anti-human cytokeratin (as the primary antibody) 92 and Alexa 555 goat anti-mouse (as the secondary antibody type) 98.
  • the primary conjugated fluorophore (Alexa 488) 90 is the weaker channel while the signal of the secondary antibody conjugated fluorophore (Alexa 555) 96 is amplified.
  • FIGURE 5 depicts a procedure which uses an enzyme based signal amplification method, such as the Tyramide Signal Amplification (TSA (trade mark)) to amplify the secondary antibody (e.g., Alexa 555).
  • TSA Tyramide Signal Amplification
  • Tyramide Signal Amplification involves using an enzyme to catalyze a chemical reaction that amplifies the signal.
  • a non-conjugated primary antibody 100 bound to a cell of interest 102 is used, but instead of regular dye-conjugated 2 nd antibody, a 2 nd antibody 104 conjugated to an enzyme horse radish peroxidase (HRP) 106 is used to target the primary antibody 100.
  • HRP horse radish peroxidase
  • a reagent 108 containing a marker 110(*), such as a fluorophore dye (e.g. , Alexa 555), hydrogen peroxide H 2 O 2 112( ⁇ ) and a tyramide mixture 114( ⁇ ) in a non-reactive form is applied.
  • a marker 110(*) such as a fluorophore dye (e.g. , Alexa 555), hydrogen peroxide H 2 O 2 112( ⁇ ) and a tyramide mixture 114( ⁇ ) in a
  • the present procedure takes advantage of the amplification concepts obtainable via use of TSA to create an asymmetric dye concentration by incorporating the other dye (e.g., Alexa 488) 116 in the form of a conjugate to other secondary antibody 104, at a level different from dye 110, and in a particular embodiment, the amount of dye 116 would be less than the amount of dye 110.
  • the other dye e.g., Alexa 488
  • TSA has been cited as the amplification technique, other known biological amplification techniques may also be used.
  • FIGURE 6 shown is a further procedure to increase the spectral resolution of a sample.
  • a first type (primary) antibody 120 is, again, bound to a cell of interest 122 using known techniques.
  • This procedure then uses a single fluorophore dye 124 which is associated with a 2 nd type (secondary) of antibody 126, which itself is configured to target a 1 st antibody 120.
  • Single dye 124 is excited in a non-conventional manner where the emission is collected and used in a novel way.
  • dye 124 may be Alexa 555, and the Alexa 555 conjugated antibody 126 is excited by a 488nm laser. Since its excitation efficiency with a 488nm laser is only 14.2% (see Table 1), slightly higher than normal concentrations of Alexa 555 are used.
  • a 150x dilution is used to compensate for the loss in excitation efficiency.
  • the dilution is the dilution of an antibody-dye (or marker) in a buffer (such as phosphate buffered saline) to be applied onto the sample under investigation. More particularly, the dilution is in a range of six to seven times less than the accepted industry standard of approximately 1000x dilution.
  • fluorescence of these imaging events may be undertaken using a dye with a lower emission wavelength and collected at a shorter emission wavelength (e.g. 525nm when using 488nm laser) where most autofluorescence occurs. Together with the emission at longer wavelength (e.g. 585nm for Alexa 555 dye) the ratio of emissions at the two wavelengths using a two-dye system provides an additional distinctive set of data for filtering out false positives.
  • FIGURE 7 illustrates the absorption curve 130 for the DyeMer 488/630 dye, which shows it excites well at 488nm besides its main absorption peak around 615nm; but has most of its emission centered around a peak at 630nm, as depicted by emission curve 150.
  • Tandem dyes are conjugates of two dyes, with the emission of the first dye being absorbed by the second dye.

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Claims (10)

  1. Verfahren zum Durchführen eines laserbasierten Scanverfahrens an einer Probe, die eventuell interessierende Zellen enthält, wobei die Probe mit einem Verfahren aufbereitet wird, das Folgendes umfasst:
    Platzieren eines ersten Materials, das eventuell die interessierenden Zellen enthält, auf einem Objektträger,
    Zugeben von mindestens einer ersten Art von Antikörper, die eingerichtet ist, die interessierenden Zellen zu markieren,
    Zugeben einer zweiten Art von Antikörper, die eingerichtet ist, die erste Art von Antikörper zu markieren,
    Zusammenbringen von mindestens einer ersten Markierungssubstanz mit der ersten Art von Antikörper und/oder der zweiten Art von Antikörper, und
    Zusammenbringen einer zweiten Markierungssubstanz mit der ersten Art von Antikörper und/oder der zweiten Art von Antikörper,
    wobei die Menge der ersten Markierungssubstanz und die Menge der zweiten Markierungssubstanz nicht gleich sind,
    wobei die erste Markierungssubstanz und/oder die zweite Markierungssubstanz gewählt wird bzw. werden, um eine suboptimale Anregung für die Wellenlänge eines ausgewählten Lasers des laserbasierten Scansystems aufzuweisen, und sich die Emissionswellenlänge der ersten Markierungssubstanz von der der zweiten Markierungssubstanz unterscheidet,
    ein Strahl Laserlicht über die Probe bewegt wird und Lumineszenz, die von der Probe von jeder der Markierungssubstanzen emittiert wird, erfasst und verglichen wird,
    die erfasste Lumineszenz mit den Positionskoordinaten des Strahls korreliert wird und die erfassten Bildinformationen verarbeitet werden, um falsch positive Bildereignisse von richtig positiven Bildereignissen zu filtern, und
    die Probe anschließend mit einem Fluoreszenzmikroskop an einer Stelle, die mit einem positiven Bildereignis korreliert, bildlich dargestellt wird.
  2. Verfahren nach Anspruch 1, wobei eine Vielzahl der zweiten Art von Antikörper vorhanden ist, die erste Markierungssubstanz mit einer ersten Untergruppe der Vielzahl zusammengebracht wird und die zweite Markierungssubstanz mit einer zweiten Untergruppe der Vielzahl zusammengebracht wird.
  3. Verfahren nach Anspruch 2, wobei die erste und zweite Untergruppe der Vielzahl um die Bindung an die erste Art von Antikörper konkurrieren.
  4. Verfahren nach Anspruch 1, wobei die erste Markierungssubstanz mit der ersten Art von Antikörper zusammengebracht wird und die zweite Markierungssubstanz mit der zweiten Art von Antikörper zusammengebracht wird.
  5. Verfahren nach Anspruch 1, wobei die erste Markierungssubstanz durch Konjugation des Antikörpers an ein Enzym mit dem Antikörper zusammengebracht wird, wobei das Enzym in der Lage ist, einen Bestandteil, der die Markierungssubstanz umfasst, optional in Gegenwart weiterer Reagenzien aus einem nicht reaktionsfähigen in einen reaktionsfähigen Zustand umzuwandeln, und das Verfahren das Kontaktieren der Probe und des Antikörper-Enzym-Konjugats mit dem nicht reaktionsfähigen ersten Markierungssubstanzbestandteil, wenn erforderlich, in der Gegenwart der weiteren Reagenzien, umfasst, wodurch der erste Markierungssubstanzbestandteil in seinen reaktionsfähigen Zustand umgewandelt wird und sich in der Nähe des Antikörper-Enzym-Konjugats absetzt.
  6. Verfahren nach Anspruch 1, bei dem der Laser eine Wellenlänge von 488 nm hat.
  7. Verfahren nach einem vorhergehenden Anspruch, bei dem die Lumineszenz bei 525 nm ± 10 nm und bei 580 nm ± 10 nm erfasst wird.
  8. Verfahren nach einem vorhergehenden Anspruch, bei dem der Bereich der Probe, der von dem Laserstrahl bestrahlt wird, einen Durchmesser im Bereich von 1 bis 20 µm aufweist.
  9. Verfahren nach einem vorhergehenden Anspruch, bei dem der Strahl über die Probe gerastert wird, indem der Strahl in einem beweglichen Reflektor reflektiert wird und die Probe relativ zum Reflektor bewegt wird.
  10. Verfahren nach einem vorhergehenden Anspruch, bei dem die Lumineszenz von einer Gruppe von Lichtwellenleitern aufgenommen und von den Leitern an einen Detektor weitergeleitet wird, wo sie erfasst wird.
EP07100507A 2006-01-17 2007-01-12 Neue Verfahren zur Immunozytfärbung zur verbesserten Unterscheidung des Färbungsverhältnisses bei der Erkennung seltener Ereignisse Not-in-force EP1808689B1 (de)

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